Stabilization of animal and poultry feeds containing a tetracycline antibiotic



United States Patent 3,427,166 STABILIZATION OF ANIMAL AND POULTRY FEEDS CONTAINING A TETRACYCLINE ANTIBIOTIC Anthony Abbey, Jamesburg, Robert Berendt Fortenbaugh,

Gladstone, and Irving Klothen, Princeton, N.J., assignors to American Cyanamid Company, Stamford, Conn., a corporation of Maine No Drawing. Filed Jan. 21, 1964, Ser. No. 339,088 U.S. Cl. 992 11 Claims Int. Cl. A231; 1/175 ABSTRACT OF THE DISCLOSURE Stabilizing animal and poultry feeds containing an edible animal feedstuff and dried fermentation harvest mash solids containing a tetracycline antibiotic against losses in potency for extended periods of time by adding said fermentation mash and prior to harvest thereof, an effective amount of aluminum ion.

This invention relates to the stabilization of animal and poultry feed supplements and to animal and poultry feeds containing a tetracycline type antibiotic and more particularly is concerned with a novel process of preparing animal feed supplements and/or animal feeds which are stable upon prolonged storage against loss of antibiotic potency.

In recent years the use of antibiotics in animal feeds for improving growth characteristics and efiiciency of feed utilization has become of considerable economic importance. One of the best of such procedures is described in the United States patent to Jukes No. 2,619,420 which involves the addition to animal feeds of the antibiotics produced by fermentation of an aqueous nutrient medium with the microrganism Streptomyces aureofaciens. In practice, the fermentation mash solids are harvested, after the fermentation has been continued for the requisite length of time, and dried. The dried harvest mash solids containing the antibiotic, usually chlortetracycline, can be used as an animal feed supplement or the solids may be blended with conventional animal feed materials to produce a nutritionally balanced growth-accelerating animal feed.

The use of the dried chlortetracycline-containing fermentation harvest mash solids as an animal or poultry feed supplement has from the beginning presented very vexing problems particularly the loss of potency of the antibiotic in the feed or feed supplement upon prolonged storage, particularly under conditions of relatively high temperature and humidity. This 1055 of antibiotic potency, and which frequently runs as high as 20% over a oneyear storage period, has heretofore necessitated the addition of a suitable overage of dried harvest mash antibioticcontaining solids to the animal feed blend so as to maintain the antibiotic potency during storage. Such a procedure is unsatisfactory and among other things results in higher than desired manufacturing costs for these animal feed supplements.

Other methods for stabilizing feeds containing tetracycline antibiotics have been suggested. For example, it has been suggested to add the biologically inactive antibiotic 7chloro-4-epi-tetracycline to the supplement to stabilize it against loss of potency. It has also been suggested that calcium hydroxide be admixed with the supplement and the mixture pelletized before adding it to the feed. While such methods have met with considerable success, they require additional processing steps, pelletizing equipment or preparation of rather expensive additives.'Furthermore, to date, no entirely satisfactory method has Patented Feb. 11, 1969 been proposed whereby the supplement may be rendered stable by treatment during the preparation of the supplement itself prior to harvesting of the fermentation mash.

In commercial practice, a typical procedure for obtaining the dried harvest mash antibiotic-containing solids is carried out by harvesting the fermentation mash at the prevailing pH, that is about pH 68. A filter aid such as diatomaceous earth is then added and the solids are collected by filtration and dried. The dried filter cake containing chlortetracycline may be marketed as such or may be blended with conventional poultry or animal feed materials to produce a nutritionally balanced growthaccelerating antibiotic-containing animal feed. While this harvested product is stable, when it is admixed with feed and stored under normal conditions for prolonged periods a loss of potency occurs.

The present invention is based on the surprising discovery that if aluminum ion is added to the fermentation mash prior to harvest and the harvesting is thereafter carried out in a conventional manner, the resulting dried harvest mash solids containing the antibiotic are stable against losses in antibiotic potency for extended periods of time whether or not it is in the usual form of a dried cake or whether it is blended with conventional poultry or animal feed materials. Thus, the present invention provides for the first time a simple one-step process of stabilizin g not only the animal feed supplement but the finished feed as well against losses in antibiotic potency. It is not known why this treatment imparts such striking stability to the feed supplement even when blended with conventional feed materials but it is believed that the aluminum binds the tetracycline type antibiotic in some physical or chemical manner rendering it stable against losses in potency.

To achieve the desired stabilization, aluminum ion may be supplied from any convenient source of organic or inorganic aluminum salt which is soluble, at least to some extent, in the aqueous fermentation liquor. While aluminum chloride and aluminum sulfate are preferred, aluminum acetate, aluminum bromide and the like may be used.

Generally, between about 0.6% and 5.0% of an aluminum salt based upon the volume of wet mash has been found effective in stabilizing the antibiotic supplement. However, greater or lesser quantities of aluminum salt may be used but with less advantage, and it has been found that the most effective results are obtained when from about 1.25% to about 2.50% of an aluminum salt is employed. While it has been found that addition of from 0.6% to 5.0% of aluminum salt generally produces an adjustment of the pH of the fermentation mash to the desirable value of from 2 to 4, where such pH adjustment is not achieved, a suflicient amount of an appropriate acid or base may be added.

It has also been observed that if the mash is heated following the addition of aluminum ion and subsequent neutralization, and before harvest, to a temperature of between about -100" C. for several minutes, usually at least five, additional stability is obtained in the feed supplement and in the finished feed.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 In the following tests the chlortetracycline control mash was prepared in accordance with conventional fermentation procedures such as described in United States Patent to Duggar No. 2,482,055, and using Streptomyces aureofaciens strain A-377. The chlortetracycline test mashes were prepared by withdrawing representative ml. samples from the fermenttaion vats upon completion of fermentation and prior to harvesting. These 10v ml. samples were placed in beakers and the desired amount of aluminum salt in 20 ml. of water was admixed therewith. The pH value of these mixtures was generally found to be between pH 3.2 -3.5. However, in those instances where this 5 pH range was not achieved, a suitable acid or base was TABLE 1 Preparation of Supplement by Fermentation Process and by Instant Inventiou-Filtered and Dried Microbioassay Test Tube Con- Prep. of Samples tents, mcg. OTC/Test Tubefor TestsFilter Plain and With Poultry Feed Cake Resuspended Added After EtfiPgys Storage Ml. Mash Additive Heat, Filter Aid in 130 ml. H2O, at 5 C./min. ml./test tube Plain, No Feed Added, Feed Added 10 g. per test tube 100 m1. GTO None None Celite 512 13 70, 000 23, 500 100 m1. T0 None, adj. to pH 90100/5 ---do 13 58,000 23,250

3.2 with H01. 100 ml. CTC None 90-1 13 70,500 32,200 100 ml. CTC 1.25% AlCls None 13 49, 150 40, 250 100 m1. OTC 13 45, 250 42, 700 100 ml OTC 1 25% A1013. 13 49, 750 48, 250 100 m1 CTC 1 25% A1013" 13 55,250 49, 500 100ml OTC 1.6% A1z(SO4)3 13 56,000 50,750

added to adjust the pH to Within the desired range of pH 2-4. A sufiicient amount of sodium hydroxide solution was then added to each mixture to adjust the pH thereof to a value of from 7.0 to 7.2. Then with continued stirring, the mixtures were heated to from 90 C.100 C. and maintained at such temperature for about five minutes. The mixtures were cooled to about 25 C. and, where necessary, the pH readjusted to about 7 .2. Approximately 1.5 grams of Celite No. 512 filter aid was introduced into the cool mixture and the entire mixture filtered through Whatman No. 1 filter paper previously coated with 1.5 grams of Celite No. 512. The filtrate was discarded and the filter cake transferred to a Petri dish and dried in a vacuum oven for 18 to 22 hours at about 48 C.-55 C. and 25 inches of vacuum. The dried filter cake was then ground to a fine uniform powder, resuspended in water to original treatment volume (130 ml.) and approximately 13 ml. 5 original mash volume) from each treatment was placed in a number of test tubes. Control samples for each treatment were simply capped and stored without the addition of feed thereto. However, to substantially identical samples from each treatment were added grams of a commercially available poultry feed. An example of feed used, contained a minimum of crude protein, minimum crude fat 4%, and maximum crude fiber 5% and contained the following ingredients:

Riboflavin supplement D-activated animal sterol Vitamin A oil Meat and bone scrap Soybean oil meal Dehydrated alfalfa meal Corn meal Hominy feed Wheat standard middlings Feeding cane molasses Low fluorine rock phospate, 2% Calcium carbonate, 2% Manganese sulfate, .0125 Calcium pantothenate Niacin Animal fat (preserved with butylated hydroxyanisole) All samples were stored at 56 C. for three days, then removed from storage and assayed microbiologically for chlortetracycline content.

From the data provided in Table 1 above, it is evident that:

(1) Admixture of animal or poultry feed with chlortetracycline-containing supplements produces instability and acute loss of potency of the antibiotic under stress conditions at 56 C.;

(2) Chlortetracycline-containing feed supplements prepared in accordance with the instant invention are significantly more stable in the presence of animal or poultry feeds than those prepared by conventional procedures, e.g., without addition of aluminum ion and/or heat;

(3) Heating of the mash prior to harvesting produces increased stability of the mash regardless of whether the mash is produced by the conventional process or by the process of the instant invention;

(4) The stability of the supplement achieved by use of aluminum ion in the preparation thereof is augmented by the use of heat in the preparation prior to harvesting.

EXAMPLE 2 Following the procedure set forth in Example 1, chlortetracycline, oxytetracycline and tetracycline antibiotic feed supplements were prepared with various concentrations of aluminum chloride used in the fermentation treatment prior to harvesting of the mash. The filter cakes obtained were dried, ground and resuspended in water. For the accelerated test system, 13 ml. samples from each treatment were placed in test tubes and the test tubes capped and stored at 56 C. Likewise, similar 13 ml. samples from each treatment were placed in test tubes with 10 grams of poultry feed as used in Example l. These samples were capped and stored with the others at 56 C. for three days and thereafter all samples were removed and assayed microbiologically for antibiotic content.

The results obtained are provided in Table 2 below, where it can be readily seen that in these accelerated tests, the antibiotic-containing supplements prepared by conventional methods were unstable when stored in admixture with feed and water. Additionally, it can be seen that all supplements prepared by the process of the instant invention were very stable even when stored under the most adverse conditions of the test. It can be further noted that the use of 1.25% to 2.5% of AlCl in the preparation of the various supplements produced maximum stability of the supplement.

TABLE 2 Mierobioassay Test Tube Con- Prep. oi Samples tents,mcg. antibiotic/Test Tube- !or Tests-Filter Plain and With Poultry Feed Cake Resuspendtgd Added Alter; 261233 5 Storage Ml. Mash Additive Heat, Filter Aid in H20, mL/te C./min. tube Plain, No Feed Added, Feed Added 10 g. per test tube 00 ml. 90-100 5 13 as, 000 8,500 100 ml. 9 100/5 13 26, 500 24, 625 100 ml. 90-100/5 13 42, 250 25, 225 100 m1. 90-100/5 13 34, 375 27, 850 100 ml. one 100/5 13 11, 325 3, 375 100 ml. 0 5% AlCl: 9 100/5 18 13, 300 8, 075 100 ml. 90-100/5 13 15, 400 12, 150 100 ml. Q 100/ 5 13 14, 925 12, 250 100 ml. 100/ 5 13 11, 650 5, 600 100 ml. 90-100/5 13 10, 500 6, 100 100 ml. 90-100/5 13 9, 325 8, 150 100 1111. 90-100/ 5 13 8, 325 8, 750

1 Chlortetracycline. 9 Oxytetracycline. I Tetracycline.

EXAMPLE 3 A process according to claim 1 m which an aluml- Following the procedure set forth in Example 1, four chlortetracycline feed supplements or mashes were prepared and tested. Comparative data obtained by microbiological assay of each supplement is provided in Table 3 below.

From these data it is evident that the addition of AlCl to mash prior to harvesting produces increased stability of the mash. It also shows that commercial filter aids, other than Celite may be used successfully in the preparation and recovery of the mash.

num salt is used as the source of aluminum ion and in which the salt is present to the extent of between about 0.55% based on the weight of the fermentation mash.

3. A process according to claim 1 in which the tetracycline antibiotic is chlortetracycline.

4. A process according to claim 1 in which the tetracycline antibiotic is tetracycline.

5. A process according to claim 1 in which the tetracycline antibiotic is oxytetracycline.

TABLE 3 Preparation oi Supplement by Fermentation Process and by Instant Invention-Filtered and Dried Microbloassay Test Tube Con- Prep. 01 Samples tents, meg. CTC/Test Tubeior Tests-Filter Plain and With Poultry Feed Cake Resuspended Added Altert igfilggys' Storage Ml. Mash Additive Heat, Filter Aid in H1O, mlJtest C./min. tube Plain, No Feed Added, Feed Added 10 g. per test tube 100ml. GTO None 90-100/5.-. 3% Cellte 512 13 67,750 34,250 100ml. CIC 1.25% AlCla 90100=5 do 13 60,250 51,500 100 ml. CTC 1.25% A101: 90100=5--- 2.4% Dicalite 436--.- 13 64, 500 56,000

EXAMPLE 4 6. A process according to claim 1 in which the tetra- Following the procedure of Example 3 and employing similar ratios of ingredients, but on a pilot scale, essentially identical feed supplements were prepared. These supplements were assayed microbiologically and the data obtained is given in Table 4 below.

From the data it can be seen that supplements prepared on a pilot scale are essentially similar to those obtained with preparations on a smaller scale.

TABLE 4 Pilot Plant Preparation of Supplement by Fermentation Process and by Instant Invention-Filtered and Dried Microbioassay Test Tube Con- Prep. 01 Samples tents, mcg. OTC/Test Tubeior TestsFilter Plain and With Poultry Feed Cake Resuspeuded Added AfterttgfiDgys' Storage Mash Additive Heat, Filter Aid in H0, mlJtest "(l/min. tube Plain, No Feed Added, Feed Added 10 g. per test tube 100 m1. CTC mash taken None 90-100/5... 3% Celite 512..-..- 13 63, 750 25,300

from vats for test purposes.

D0 1.25% A1013.-." 90-100/5 do 13 58,500 50,750 D0 1.25% A1013.-. 90-100/5 --d0 13 63, 250 48, 750

We claim: said fermentation mash solids and adding said mash to 1. A process for the production of a dried fermentation harvest mash solids animal feed supplement containing a tetracycline antibiotic which comprises adding to a fermentation mash containing said antibiotic and prior to harvest thereof an effective amount of aluminum ion in a quantity sufficient to stabilize the antibiotic against losses in potency for extended periods of time when the supplement is incorporated into a major amount of an edible animal feedstufl.

a major amount of an edible animal feedstuif whereby said feedstuif will be stable against losses in potency for extended periods of time. i

8. A process according to claim 7 in which the antibiotic is chlortetracycline.

9. A process according to claim 7 in which the antibiotic is tetracycline.

10. A process according to claim 7 in which the antibiotic is oxytetracycline.

7 8 11. A process according to claim 7 in which the -anti- FOREIGN PATENTS biotic is demethylchlortetracycline. 548,652 11/1957 Canada 814,671 6/ 1959 Great Britain. References Cited UNITED STATES PATENTS v 5 RAYMOND N. JONES, Primary Examiner. 2,847,471 8/1958 Vandeputte et a1. 195-80 X KLARE Assistant Emmi 2,878,289 3/1959 McCormick et a1 99-2X US Cl 3,122,578 2/1964 Remmers et a1 167-6SX 3,157,512. 11/1964 Muller 99-2X 99-4; 424-227 

